Fig. 2.

Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the GT activity of HCE and RNA cap formation. (A) CTD phosphorylation by P-TEFb/Tat stimulates the GT activity of HCE. HCE was incubated with [α-³²P]GTP in the absence or presence of bead-bound RNAP II. Guanylylation of HCE was assessed by SDS/PAGE followed by autoradiography. (B) Ser-2 phosphorylation does not influence the effect of CTD Ser-5 phosphorylation on HCE activation. Guanylylation of HCE was performed by incubating HCE with [α-³²P]GTP in the absence or presence of bead-bound GST-CTD and then resolved by SDS/PAGE followed by autoradiography. (C) CTD phosphorylation by P-TEFb/Tat stimulates RNA cap formation. A T7 polymerase run-off RNA (72-mer) was incubated with HCE and [α-³²P]GTP in the absence or presence of bead-bound RNAP II. GMP-labeled RNA was then analyzed by electrophoresis through 10% polyacrylamide gels containing 7 M urea in TBE buffer followed by autoradiography. (D) CTD Ser-2 phosphorylation does not influence the effect of CTD Ser-5 phosphorylation on RNA cap formation. Transfer of GMP was performed by incubating RNA with HCE and [α-³²P]GTP in the absence or presence of bead-bound GST-CTD. GMP-labeled RNA was then analyzed by electrophoresis through 10% polyacrylamide gels containing 7 M urea in TBE buffer followed by autoradiography.